Methods and apparatus for sensing a physical substance

ABSTRACT

Methods and apparatus for sensing a physical substance, in which the physical substance is positioned in close proximity to a first surface of at least one surface plasmon enhanced illumination apparatus. The first surface is irradiated with electromagnetic radiation, and a change in a resonance condition of the at least one surface plasmon enhanced illumination apparatus due to the physical substance is detected.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Non-provisional patentapplication Ser. No. 10/218,928, filed on Aug. 14, 2002, entitled“Surface Plasmon Enhanced Illumination System.” Ser. No. 10/218,928 inturn claims the benefit of U.S. Provisional Application Ser. No.60/312,214, filed on Aug. 14, 2001. Each of the foregoing applicationsis incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to methods and apparatus in which target areasare illuminated with one or more spots or lines of light having verysmall dimensions and the use of these spots or lines of light andchanges to them as a sensing technique.

BACKGROUND OF THE INVENTION

Typical optical microscopy, far-field light microscopy, cannot resolvedistances less than the Rayleigh limit. The Rayleigh criterion statesthat two images are regarded as just resolved when the principal maximum(of the Fraunhofer diffraction pattern) of one coincides with the firstminimum of the other [see Born, M. and Wolf, E. Principles of Optics.Cambridge University Press 6^(th) ed. p. 415 (1980)]. For a circularaperture, this occurs at $w = {0.61\frac{\lambda}{N\quad A}}$

For example, the wavelength (λ) at the peak emission of a greenfluorescent protein (EGFP) is 508 nm. Hence, for a very high numericalaperture (NA) of the objective, NA of 1.4, the minimum separation (w)that can be resolved in a GFP labeled sample is 221 nm. Currently, thereare several possible methods for achieving resolution of spatiallocations of proteins below the Rayleigh limit. They include: ConfocalMicroscopy, Fluorescence Resonance Energy Transfer (FRET), Atomic ForceMicroscopy (AFM), Near-Field Scanning Optical Microscopy (NSOM),Harmonic Excitation Light-Microscopy (HELM), Stimulated EmissionDepletion Microscopy (STED-Microscopy) and Electron MicroscopeImmunocytochemistry.

Confocal Microscopy is a technique in which a very small aperture(s)is/are placed in the optical path to eliminate any unfocused light. Thisallows for a substantial increase in signal to noise ratio overconventional light microscopy. Also, it is possible to reduce the widthof the central maximum of the Fraunhoffer pattern using a small slit oraperture. This, in turn allows a substantially enhanced resolution of1.4 times better than the Rayleigh limit. Therefore, with this method,using the above protein as an example, a spatial resolution of 156 nm isachieved.

Typical confocal microscopy is not without disadvantages. By increasingthe signal to noise ratio by decreasing the aperture size, the totalsignal level decreases concurrently. To bring the signal back to auseful level, the input power level must be increased. This, in turn,not only can cause photo-bleaching in the fluorophores at which oneintends to look but also the surrounding area where the light isincident, just not collected. A method around this is to use two-photonexcitation. Fluorescence from the two-photon effect depends on thesquare of the incident light intensity, which in turn, decreasesapproximately as the square of the distance from the focus. Because ofthis highly nonlinear (.about.fourth power) behavior, only those dyemolecules very near the focus of the beam are excited, while thesurrounding material is bombarded only by comparatively much fewer ofthe low energy photons, which are not of enough energy to cause photobleaching. Multi-photon excitation requires highly skilled techniciansand is somewhat expensive for clinical use. Because it acquires only asmall area at once, the surface must be scanned in three dimensions formapping.

Fluorescence Resonance Energy Transfer (FRET) can provide exquisiteresolution of single chromophores. The resonance occurs when onefluorophore in an excited state transfers a portion of its energy to aneighboring chromophore. For this to take place, there must exist someoverlap between the emission spectrum of the fluorophore to absorptionspectrum of the chromophore (the frequency of the emission spectrumshould be somewhat higher than the absorption spectrum of thechromophore). The process does not occur through photonic emission andabsorption but through a dipole-dipole interaction. The strength of theinteraction varies as r⁻⁶. The Forster distance [see Forster, T Discuss.Faraday Soc. 27 7-29 (1959)] is the fluorophore loses energy toradiative decay or dipole-dipole interaction. The Forster distance,essentially, is the threshold at which FRET will no longer exist for agiven pair. Typically the Forster distance is between 3 and 6 nm [seePollok & Heim “Using GFP in FRET-based Applications” Trends in CellBiology 9 pp 57-60 (1999)].

By placing either of the complementary pair near the other, resolutionsof less than the Forster distance can be attained. The problem with thistechnique in determining relative locations is that one of the pairneeds to be located within the resolution tolerances desired for spatialmapping. This can be achieved by placing one of the pair on a probe usedin either atomic force microscopy (AFM) or near-field scanning opticalmicroscopy (NSOM). Another problem is that dipole-dipole interactionsare dependent on the relative orientation of the two. To maximize signalfrom the interaction would require a 3D scan around one of the pair.

Atomic Force Microscopy (AFM) can be envisioned as a very small (usuallymetal) stylus dragged across a surface giving feedback as to the height,Z, of the stylus relative to the surface. Resolution can be as fine asthe scanning step size (typically 5 nm). By scanning across the surface,X and Y coordinates are obtained provided that the origin remains fixed(i.e., that there is no drift in the translation stage due to thermal orother effects). There are many methods for ensuring that the stylus doesnot actually contact the sample but maintains very accurate resolutionof the Z coordinate. Because only surface morphology is measured,differentiating several molecules can be extremely difficult unless thedimensions and orientations of those molecules are well known. Asolution to this might be to add tags of discrete lengths or shapes,which could be bound indirectly to the molecules of interest. Thismethod, however, would require that the tissue sample to be planarbefore the tags were bound to the surface.

To increase the information of AFM, one could use Near-Field ScanningOptical Microscopy (NSOM or SNOM). NSOM uses a principle similar to AFMin which a stylus is scanned over a surface providing topographicalinformation. However, the stylus is a conductor of photons. By emittinglight from the tip of the stylus, optical measurements such asfluorescence can be obtained. Most often, these styli are fiber probesthat have tapered tips and then are plated with a conductive material(aluminum is most often chosen as its skin depth for optical radiationis quite low, .about.13 nm at 500 nm) with a small aperture where thecoating is broken. [See Betzig & Trautman “Near-Field Optics:Microscopy, Spectroscopy, and Surface Modification beyond theDiffraction Limit” Science 257 pp 189-195 (1992)]. Another approach isto use what are called “apertureless probes” [see Sanchez, Novotny andXie “Near-Field Fluorescence Microscopy Based on Two-Photon Excitationwith Metal Tips” Physical Review Letters Vol 82 20 pp 4014-4017 (1999)]where an evanescent wave is excited by bombardment with photons at thetip of a sharpened metal probe. Because the tip can be made very sharp(radii of 5 nm are achievable), resolutions can be correspondinglysmaller. An associated problem with the “apertureless probes” is thatthe probe generates a white light continuum, which significantlydecreases the signal to noise ratio.

By making the diameter (assuming a circular geometry) of the emissionportion of the tip of the stylus very small (smaller than resolutiondesired) and keeping the tip to sample distance less than that distance,so that the diffraction is small, a nanometric light source isavailable. This light source can be used to excite fluorescence in thesample. Because the size of the source is very small and the scanningincrements are also very small, highly resolved information on spatiallocations of the fluorophores can be gleaned by inspection in the farfield. Alternatively, the probe can be used for collection, measuringfluorescence or reflection or even transmission from illumination fromthe other side of the sample.

Because the aperture size in a conventional probe is so much smallerthan the wavelength of the excitation light and only an evanescent modeis supported, very little light is transmitted through the aperture.Diffraction effects limit the effective collimated length from theaperture to less than diameter of the aperture. This, then, requiresthat the aperture be held below a maximum height above the surface ofthe sample. Ideally, a fixed height above the surface (usually less than10 nm) is used for relative contrast measurements. The height of theaperture relative to the surface is kept constant by measuring the shearforce on the tip of the probe or by optical methods and is modulated tomaintain that height. For this reason, NSOM is particularly susceptibleto vibrations and experimental work requires isolation platforms.

Scanning the surface takes a fair amount of time. Thermal drift incommercially available open and closed loop nanometric scanning stagesis about 20-30 n/min. [see Frohn, Knapp and Stemmer “True opticalresolution beyond the Rayleigh limit achieved by standing waveillumination” Proceedings of the National Academies of Science Vol. 97,13 pp 7232-7236 (2000)]. This can be severely limiting if scanning timeis more than a few tens of seconds and resolution less than 50 nm isdesired. If the surface is scanned for several different types ofmolecules, the required time to investigate a single cell becomes fartoo large for use in a clinical setting and would require multiplehomings of the scanning stage. An approach to diminishing the scanningtime may be to scan with multiple probes concurrently. This approachwould be limited to just a few probes as on a small (20² μm²) surface,the relatively large size of the probes' bodies would interferemechanically with each other.

U.S. Pat. Nos. 5,973,316 and 6,052,238 issued to Ebbesen et al. of theNEC Research Institute, Inc. describe a NSOM device which employs anarray of subwavelength apertures in a metallic film or thin metallicplate. Enhanced transmission through the apertures of the array isgreater than the unit transmission of a single aperture and is believedto be due to the active participation of the metal film in which theaperture array is formed. In addition to enhancing transmission, thearray of apertures reduces scanning time by increasing the number ofnanometric light sources.

A second method for increasing the number of light sources illuminatesthe sample with a mesh-like interference pattern and by post processingof the images. In Harmonic Excitation Light Microscopy (HELM), a laseris split into four beams and two of those beams modulated to produce anextended two-dimensional interference field with closely spacedantinodes. By introducing the beams at an angle to the surface to beimaged, an effective offset in reciprocal space is produced around anorigin. If four images are taken around this origin and one at theorigin, it is possible to construct, with post processing, a smallersingle antinode which acts as a nanometric light source. This processcan result in a lateral resolving power of close to 100 nm or half ofthe Rayleigh distance for green light. Because only a few images arerequired to map an entire surface, the acquisition time is extremelyshort (around 1.6 s for a 25 μm×25 μm area with 100 nm resolution.) Dueto the required precision in the location of the four images around theorigin and the drift associated with the scanning stage, it is unlikelythat the resolution will be dramatically increased.

Another new form of microscopy is that introduced by Klar et al. [seeKlar, Jakobs, Dyba, Egner and Hell “Fluorescence microscopy withdiffraction resolution barrier” Proceedings of the National Academies ofScience Vol 97 15 pp 8206-8210 (2000)] called Stimulated EmissionDepletion (STED) Microscopy. STED microscopy is based on a method ofquenching fluorescence by stimulated emission depletion reducing thefluorescing spot size. [See Hell & Wichmann “Breaking the DiffractionResolution Limit by Stimulated-Emission-Depletion FluorescenceMicroscopy” Opt. Lett 19 11 780-782 (1994); Lakowicz, Gryczynski,Bogdanov and Kusba. “Light Quenching and Fluorescence Depolarization ofRhodamine-B and Applications of this Phenomenon to Biophysics” J. Phys.Chem. 98 1 334-342 (1994); Hell, S. W. Topics in FluorescenceSpectroscopy, ed. Lakowicz (Plenum Press, NY), Vol. 5, pp. 361-422; andKlar & Hell “Subdiffraction resolution in far-field fluorescencemicroscopy” Opt. Lett 24 14, 954-956 (1999)]. Fluorescence can bequenched by subjecting a fluorophore to light at the lower energy edge(red side) of its emission spectrum. This forces the fluorophore to ahigher vibrational level of the ground state, which, by decay of thatstate prevents re-excitation. Fluorescence can be turned on, with anordinary excitation source, and turned off, with the STED beam, at will.By introducing an interference pattern in the STED beam, a local set ofmaxima and minima can be created. If the maxima of the STED beam areoverlaid onto the fluorescence induced by the excitation beam, thefluorescence is quenched. However, where the minima occur, fluorescencecontinues. The fluorescing spot size is controlled by the union of theminimum or minima of the STED beam and the maximum of the excitationbeam. Because STED is nonlinear with intensity, the sharpness of theminimum, maximum transition can be effectively increased allowing anarrow, almost delta behavior to be displayed. This, however, can resultin severe photo stress to the sample and, possibly, dual photon effects,causing competing modes in the area where quenching is desired. So far,resolution in the radial (X, Y) direction is around 100 nm, but there isno reason to expect that the resolution can't be substantially improved.Once again, though, STED microscopy is a scanning type and will sufferfrom the same drawbacks all scanning instruments do, (e.g., thermaldrift, vibration problems, registration of near field excitement withfar field collection and scan time.)

SUMMARY OF THE INVENTION

The present invention contemplates a different technique to achievesub-Rayleigh criterion resolution, which is here called “Surface PlasmonEnhanced Illumination” (SPEI). SPEI is related to NSOM in thatnanometric light sources are created by subwavelength apertures. Byapplying the principles of the present invention, a significantreduction in the size of the area illuminated by each aperture isachieved, resulting in significantly improved resolution.

The present invention takes the form of methods and apparatus thatemploy novel physical structures to provide nanometric spot or lineillumination. In accordance with the invention, one or more aperturesare formed through a first planar conductive material. Each aperture(which may be either a hole or a slit) has at least one cross-sectionaldimension which is less than the wavelength of light which is incidentto the planar material. In accordance with a feature of the invention,the structure includes means for confining the electronic excitationinduced in that portion of the planar surface near the end of theaperture from which the light exits.

The conductive plane that receives the incident light may be placed onone outer surface of a dielectric material. The dielectric material'sinterface with the conductive plane that receives the incident lightestablishes a substantially different effective dielectric function inthat interface than that of the conductive plane that receives theincident light. This difference in effective dielectric functionprevents the excitation of large densities of surface plasmons innon-illuminated plane of the metal if monochromatic light is used at theresonant wavelength of the illuminated metallic plane. Therefore lightshould not be substantially emitted from the non-illuminated metallicplane.

Alternatively, the sidewalls of the aperture may be conductive toconduct excitation currents and act as a pseudo-waveguide for the lighttraveling through the aperture. At the exit end of the aperture, theamount of exposed conductive material is limited to an area immediatelysurrounding the hole exit by a dielectric material, or by a groove cutinto the surface of the conductive material at the exit plane to a depthsubstantially deeper than the skin depth of the induced excitation andof such width and spacing to prevent an unwanted resonance of surfaceplasmons in that surface.

Alternatively, the conductive plane that receives the incident light maytake the form of a “good metal” layer with a “bad metal” layer havingsignificantly different dielectric properties being sandwiched betweenthe good metal layer and a dielectric substrate. The bad metal layer ispreferably opaque to the light to be emitted from the surface of thegood metal and its resonance (as determined by its dielectric function,the surface roughness and the dielectric functions of the materials oneither side of the bad metal layer) should be very different from theresonance of the “good” metal, such that at desired frequency, lighttransmitted is emitted only from the holes and not from the exit surfaceof the array. The insulating dielectric substrate ensures that there canbe no surface plasmon excitation from the good metal layer through thelight barrier. When a bad metal layer is used that is both opaque tolight and has sufficiently different dielectric properties relative tothe good metal to eliminate resonant coupling, the dielectric insulatormay be eliminated.

The present invention substantially reduces, compared to an array ofsubwavelength apertures in a monometallic film such as those describedby Ebbesen et al., the size of the area of illumination produced by eachaperture using the combination of a metallic layer on which surfaceplasmons are induced by incident light and surface composed of amaterial of substantially different dielectric function, such as aninsulator or a different metal, so that the excitation of the surfaceplasmons in the light emitting surface in the exit surface layer will bedifferent than those excited in the metallic layer that is excited bythe incident light, and only the light from the decaying resonantsurface plasmons of the exit layer will emit from that surface. Thephotons associated with the resonance of the incident or upper surfacewill be constrained to exit from the hole itself or from the walls ofthe hole.

In accordance with the invention, the light barrier comprises anilluminated surface consisting of a continuous conductive metallic layerin combination with an exit layer having substantially differentdielectric properties. One or more apertures through the barrier (one ormore holes or slits) then form “photonic funnels” through the barrier.Note that confining or eliminating electronic surface excitation on thesurface opposite to the illuminated surface works with a single apertureas well as an array of apertures.

The invention may advantageously take the form of array of apertures(holes of slits) formed in structure consisting of a dielectricsubstrate coated with a conductive metal film on one or both surfaces,or by a thick metallic film, and which further incorporates means forconfining the electronic surface excitation to an area immediatelyadjacent to the apertures where light exits the structure. The means forconfining the electronic surface excitation preferably takes the form ofa layer of material having dielectric properties that differsubstantially from those of the illuminated metal layer, and may consistof a dielectric insulator, a “bad metal” having different dielectricproperties, grooves or surface irregularities at the exit surface, or acombination of these. The structure which confines the electronicsurface excitation restricts the size of the spot or line ofillumination from each aperture, and the use of an array of apertures,or an array of surface irregularities on the metal film, increases theintensity of the illumination from each aperture

The present invention may also be applied to advantage in an opticaldata storage device. Several arrangements may be devised for combiningthe hole array with some medium for data storage. A light source, suchas a laser, may be directed onto the front surface of the hole arraywhich collects and funnels the array of light onto an optical storagemedium. The bit value stored at each position in the storage mediumcontrols the propagation of light through the storage medium to anadjacent pixel position in a charge coupled device (CCD) or other areadetectors. A translation mechanism effects movement of the storagemedium relative to the hole array in incremental steps, with each stepdistance being equal to the aperture size. In an alternativearrangement, data may be represented by illumination levels, such asgray scale values or color levels, and optical means may be used inplace of or to supplement the mechanical translation mechanism.

The well defined and highly concentrated areas of illumination createdby using such a structure as a light source provide significantadvantages in microscopy and in optical data storage devices. Theconfined illumination patterns produced in accordance with the inventionmay be used to construct a “Surface Plasmon Enhanced Microscope” (SPEM)exhibiting markedly improved resolution, to construct an optical datastorage device capable of storing larger amounts of data in opticalstorage media with much higher data access rates than is achievable withcurrent optical data storage devices, and to provide a high throughputphotolithography technique that can be applied to advantage insemiconductor fabrication and patterning for self-assembly andbiological applications.

A further embodiment of the invention provides an improved system foranalyzing one or more small objects. A radiation barrier having one ormore sub-wavelength apertures is positioned between a source ofradiation and a radiation sensor. The object to be analyzed ispositioned in the pathway of the radiation that flows through anaperture and the radiation level at the sensor is measured to evaluateone or more properties of the object. The barrier includes a conductivesurface which is illuminated by incident radiation from the source toproduce surface plasmons, and means are preferably employed to limit theextent to which surface plasmons are induced on the opposite surfaceadjacent the radiation detector, thereby focusing the light on thesensor. The presence of the object alters the radiation received at thesensor in way that may be measured to determine the property of thesmall object or objects. This technique may be employed to advantage toevaluate biological macromolecules, including protein molecules andnucleic acid molecules, as well as single cells or organisms and spores.

Means may be employed for moving macromolecules or other small object(s)to be analyzed toward and through the aperture as measurements are beingtaken. To this end, the small objects may be contained in a carrierfluid which flows through the aperture or apertures. The objects may becharged and an electrostatic field may be applied to the objects tocause them to move through the aperture, or a microfluidics system maybe used to feed a solution containing the macromolecules toward andthrough the aperture or apertures as measurements are taken. Theradiation sensor may detect changes in the intensity of the radiationcaused by the presence of the small object(s), or changes in thespectral content of the radiation may be measured to detect fluorescenceof the objects being measured, changes in the radiation pattern emittedfrom an aperture or apertures may be measured, or changes in resonancecaused by the presence of the micromolecules near the conductive surfaceof the radiation barrier may be measured. The data thus collected may beprocessed to yield information about the size, shape, orientation,fluorescence, absorbance, and transmission characteristics of theobjects being analyzed.

In a still further embodiment of the invention, a radiation barrierinterposed between a light source and a detector may be used to analyzeligands that are immobilized on the surface barrier. The ligands'binding partners bind to the ligands immobilized on the illuminatedsurface and, as that occurs, or after it has occurred, a shift inresonance or other measurable change is measured. In addition thebinding of small molecules to proteins, post translational modificationsof proteins, protein-protein interactions, and the binding of nucleicacids can all be detected.

These and other objects, features and advantages of the presentinvention may be better understood by considering the following detaileddescription of specific embodiments of the invention. In the course ofthis description, reference will frequently be made to the attacheddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross-sectional view of an aperture through a metallic film,the film being substantially thicker than the skin depth within which anoptically induced electronic excitation occurs, and the aperture havinga diameter less than the wavelength of the incident light;

FIG. 2 is a view illustrating the approximate size of the oblong-shapedarea illuminated by the light transmitted through the aperture in thefilm shown in FIG. 1;

FIG. 3 is a graph illustrating the illumination intensity in theilluminated area taken along the line 3-3 of FIG. 2;

FIG. 4 is a cross-sectional view of a thin metallic film that covers anon-metallic substrate material with an aperture through both the metalfilm and substrate having a diameter less than the wavelength of theincident light;

FIG. 5 is a view illustrating the approximate size of the circular areailluminated by the light transmitted through the aperture in structureshown in FIG. 4;

FIG. 6 is a graph illustrating the illumination intensity in theilluminated area taken along the line 6-6 of FIG. 5;

FIG. 7 is a cross-sectional view of a thin metallic film that covers thesurface of a non-metallic substrate material as well as the sidewalls ofan aperture through the substrate with the aperture having a diameterless than the wavelength of the incident light;

FIG. 8 is a view illustrating the approximate size of the circular areailluminated by the light transmitted through the aperture in thestructure shown in FIG. 7;

FIG. 9 is a graph illustrating the illumination intensity in theilluminated area taken along the line 9-9 of FIG. 8;

FIG. 10 is a cross-sectional view of a thin metallic film which covers anon-metallic substrate material, an aperture through the substrate, anda thin, annular metallic ring surrounding the aperture on the opposingsurface of the substrate, with the aperture having a diameter less thanthe wavelength of the incident light;

FIG. 11 is a view illustrating the approximate size of the circular areailluminated by the light transmitted through the aperture in thestructure shown in FIG. 10;

FIG. 12 is a graph illustrating the illumination intensity in theilluminated area taken along the line 11-11 of FIG. 10;

FIG. 13 is a cross-sectional view of a hole structure in which a thinmetallic film which covers both surfaces of a non-metallic substratematerial, and an annular notch is cut into the film at the exit surfacewhich surrounds and is spaced from the hole;

FIG. 14 is a view illustrating the approximate size of the circular areailluminated by the light transmitted through the aperture in thestructure shown in FIG. 13;

FIG. 15 is a graph illustrating the illumination intensity in theilluminated area taken along the line 15-15 of FIG. 14;

FIG. 16 is an end plan view of a multi-aperture probe constructed inaccordance with the invention;

FIG. 17 is a cross sectional view of the probe seen in FIG. 16 takealong the line 17-17;

FIG. 18 is an end plan view of an alternative structure for themulti-aperture probe constructed in accordance with the invention; and

FIG. 19 is a cross sectional view of the probe seen in FIG. 18 takenalong the line 19-19;

FIG. 20 is a cross sectional view of an alternative light barrierstructure employing “good” and “bad” metal layers;

FIG. 21 is a schematic diagram of a data storage device that uses anarray of nanometric holes to illuminate a data storage array ascontemplated by the invention;

FIG. 22 is a schematic diagram of a Surface Plasmon Enhanced Microscope(SPEM) which embodies the invention;

FIGS. 23-24 is a plan view of the location of surface patternssurrounding a central aperture used to enhance the illumination from thecentral aperture;

FIG. 25 is a schematic diagram of a flow-through sensor system foranalyzing macromolecules;

FIGS. 26-28 are side elevational views of different support structuresthat may be used to construct a sensor for analyzing macromolecules;

FIGS. 29 through 32 are intensity charts illustrating data that may beacquired by the object analysis mechanisms shown in FIGS. 25-28;

FIG. 33 is a schematic diagram of a sensor for analyzing ligands whichare immobilized at the conductive surface of a hole array and theirbinding partners;

FIG. 34 is a plan view of the location of a central square hole andsurrounding square surface irregularities that may be employed forgreater packing density; storage medium, and

FIG. 36 is a schematic diagram of a further embodiment of a data storagesystem employing the invention.

DETAILED DESCRIPTION OF THE INVENTION

As described in U.S. Pat. Nos. 5,973,316 and 6,052,238 issued to Ebbesenet al., enhanced light transmission occurs through an array of aperturesin a metal film due to the surface plasmons induced in the conductivefilm by the incident light.

FIG. 1 shows a cross section of an optically thick metal film 101. Theterm “optically thick” means that the thickness of the film 101 isgreater than two times the skin depth. For all essential purposes, thismeans that there is no direct coupling of the surface plasmons (coherentcollective excitations of electrons) at the upper surface (the interfacebetween media of index N₁ and N₂) and the lower surface (the interfacebetween media of index N₃ and N₂). In a typical case, the indices N₁,N₃, and N₄ are equal while N₂, the index of the metal film 101, issubstantially different and the metal film 101, unlike the surroundingmaterial, is a conductor of electronic charges.

If the array spacing and the dielectric functions and thickness of themetals and substrates are tailored to attain a high transmission, asignificantly higher power density than that transmitted through thesingle aperture probe used in NSOM (a ratio of about one million peraperture for a 50 nm holes) can be delivered through the apertures. Thissubstantially increases the signal to noise ratio of surface plasmonenhanced microscopy (SPEM) over the NSOM at normal resolutions and isallows a smaller hole size to be used, providing better resolution anddramatically decreasing the dwell time required for an adequate signalto be received.

Unfortunately, the coupling (indirect or direct) between the surfaces ofthe film 101 seen in FIG. 1 have effects that adversely affect desiredresolution. Sonnichsen et al., “Launching surface plasmons intonanoholes in metal films”, App. Phys. Lett. 76, 140-142 (2000) showthat, when gold, silver or aluminum films are struck with planepolarized light, surface plasmons are induced in the direction of thepolarization. When the plasmons encounter a hole, the coupling to theother side results in light emitted in a prolate shape of a majordimension of about an order of magnitude larger than the hole size. Theprolate shape is caused by the radiative decay of the surface plasmonsand is a function of the dielectric function of the metal and thewavelength of the incident light and if significant surface roughnessexists, the distance between the elements of roughness on that plane.

With a simple isotropic periodically perforated metal film, twopotential problems are encountered. First, for use in a microscope andother applications (e.g. optical data storage and photolithography)where small sources of light (high resolution) are required, theexistence of the associated prolate pattern diminishes resolution in onedimension severely. Second, the array spacing would have to be such thatpatterns did not interfere or overlap. Achieving the appropriate spacingwould in turn cause the wavelengths at which the surface plasmons areresonant to be shifted, resulting in resonant wavelengths of lowerenergy. For the excitation of commonly available fluorophores,multi-photon (probably three or four) excitation would be required. Ofcourse, the prolate pattern could simply be accepted and the resolutionin the direction of the polarization (along the major axis of thepattern) would default to that dictated by the Rayleigh criterion forthat wavelength and numerical aperture.

If a smaller spot illumination size (a nanometric light source) isrequired, the prolate shape generated from the geometry shown in FIG. 1is undesirable. If the incident light is polarized, the long dimensionof the pattern shape is probably only loosely dependent on the hole sizeand more dependent on the surface roughness, since rougher surfaces actas very small antennae, which cause SPs to decay, spatially, morerapidly than would be the case if the film surface were smooth.Moreover, the frequency of the light will also affect the pattern shape.Note also that the preferred shape of the intensity pattern for spotillumination should exhibit a step function rather than the extendedsomewhat Gaussian pattern that is seen along the major axis of theprolate shape.

In accordance with the present invention, novel structures are used tominimize or eliminate the prolate pattern described above. If theemitting surface (bottom) is no longer continuous but is insteadconstructed to constrain the propagation of surface plasmons to theimmediate vicinity of the aperture, the size of the resulting area ofillumination is significantly reduced. If the illuminated surface (top)is left as a continuous conductor with an array of circular holes in itand the bottom is segmented as described above, a photonic funnel can becreated. To minimize the effective broadening of the holes due tosurface plasmons on the bottom plane, it may be desirable to create avery sharp edge at this point in either a conducting wall or in aninsulator with less available charge to minimize anysurface-plasmons/photon interaction. It is important to note that theinsulator (in the case of a semiconductor) should have a band gapsignificantly larger than the frequency of the photons, which will bepropagating through it.

A first improved geometry for the hole array that produces a smallerillumination pattern is shown in FIG. 4 of the drawings. A thin metalconductive film 106 exhibiting the index N₂ is affixed to a substrate109 constructed of a dielectric material having the index N₃ and abandgap that is larger than the frequency of the illumination of light.The dielectric substrate 109 can be constructed of a material that istransparent (but need not be) to light at the frequency employed, suchas quartz or glass. Note that the aperture 107 need not go through thedielectric substrate if it is transparent, and such a structure may beeasier to fabricate. The substrate should have a small index ofrefraction N₃ compared to the index of the metal N₂. Note also, asdiscussed later in connection with FIG. 20, that a “bad metal” havingpoor conductivity at these frequencies (such as tungsten) may be used inplace of the dielectric 109 in combination with a “good metal”illuminated layer (such as aluminum). In fluorescence studies, ifmulti-photon excitement is employed, the bandgap should be larger thanthe sum of the photonic energies of the photons that would besimultaneously absorbed by the fluorophore. The thin layer of conductingmaterial 105 should be thicker than the skin depth of the metal at thechosen wavelengths. The geometry and composition of the heterogeneousstructure seen in FIG. 4 should be chosen so that a maximum oftransmission of illumination occurs through the hole 107 at the chosenillumination wavelength. A tunable or broad band light source may alsobe used to tune the wavelength to predetermined hole dimensions.

The advantage of the geometry shown in FIG. 4 over that presented inFIG. 1 results from the fact that there is no coupling of plasmons fromthe upper surface of the film 105 to the lower surface of the dielectricmaterial 109. This reduced coupling creates a smaller and more definedillumination pattern with steeper side slopes as illustrated in FIGS. 5and 6. It is unclear, though, what happens to the energy at the cornerinterface of the hole 107, the metal film 105 and the dielectricsubstrate 109, that is, at the boundary of the materials having theindices N₅, N₂ and N₃. If N₁, N₄ and N₅ are not all substantially equalto one (1.0), combinations of differing indices could be used to tailorthe transmission of the array apertures for a specific wavelength ormethod of illuminating the structure. For example, N, could be the indexassociated with an optical fiber, which would be coupled to a remotelight source.

A second hole array structure for reducing the size and increasing thedensity of the spot illumination is shown in FIG. 7. As before, thestructure of FIG. 7 presents at its upper surface a continuousconducting thin film metallic film 111 having the index N₂. Thestructure differs from that shown in FIG. 4 in that the metallic coatingis continued into the interior of the hole 113 as seen at 115. If thethickness of metal layer 115 in the hole interior were greater than skindepth, the effects seen in optically thick metal films as shown in FIG.1 would be duplicated from the standpoint of optical transmissionthrough the holes. However, a smaller and more concentrated output lightpattern is achieved by limiting the propagation length of SPs at theexit surface to the thickness of the film in the hole. Limiting the sizeof the excited surface area surrounding the hole exit produces aconcentrated, circular light pattern as seen in FIG. 8 rather thanprolate pattern seen in FIG. 3, thus limiting the size of the lightsource in only one of its two dimensions. As is the case with thestructure shown in FIG. 4, the indices N₁, N₄ and N₅ may be equivalentto 1 in the simplest configuration but other combinations be used totune the holes for a specific resonance. FIG. 9 graphs the steeplyskirted intensity distribution expected across the circular lightpattern along the line 9-9 of FIG. 8.

A third structure that may be used as a source of concentrated light isshown in FIG. 10. As in the structures shown in FIGS. 4 and 7, a thinmetallic film 121 covers the upper surface of a dielectric substrate123. A hole 124 through the film 121 and the substrate 123 is not linedwith a conductor as in FIG. 7. Instead, an annular ring 125 ofconductive material surrounds the exit end of hole 124 at the lowersurface of the substrate 123. The conductive ring 125 increases thecoupling with the film 124 to improve light transmission through thehole 124 but does not permit the surface excitations surrounding thehole exit to spread beyond the outer periphery of the ring 125, therebyagain achieving the more concentrated, steep skirted output lightpattern shown in FIGS. 11 and 12.

FIG. 13 shows still another structure in which a dielectric substrate127 is coated on its upper surface with a metallic film 126 and on itslower surface with a metallic film 129. The hole 128 passes through bothfilms and through the substrate and its side walls are not coated. Anannular groove seen at 130 is formed in the film 129 and surrounds andis spaced from the hole 128. The groove has a nominal outside diameterof 25 nm and inside diameter of 20 nm. The depth of the groove must beat substantially deeper than the skin depth of the material, i.e., deepenough to act as insulator with respect to induced surface excitations.The groove may have any convenient shape and may be rectangular ortriangular as well as semi-circular. Note that, by using a groove of thetype shown in FIG. 13, an optically thick metallic structure may be usedinstead of a dielectric substrate, so that the hole is effectively linedby a conductor. In both cases, the groove serves to contain the coupledelectron excitation within a surface area close to the hole exit,thereby preventing unwanted spreading of the illumination pattern. Theillumination pattern produced by the hole and groove configuration ofFIG. 13 is depicted in FIGS. 14 and 15.

As will be discussed later in conjunction with FIG. 22, the principlesof the invention may be used to construct a multi-aperture probe (MAP)which may be used to advantage in scanning microscope. FIGS. 16 and 17illustrate a MAP structure using holes with electrically conductingsidewalls of the type discussed earlier in connection with FIGS. 7 and13, while FIGS. 18 and 19 show the construction of a MAP having holeswhose sidewalls are in part non-conducting as previously discussed inconnection with FIGS. 4 and 10 of the drawings.

As also discussed above, another approach to eliminating the prolatepattern is to align the polarization with a slit. If the materialthrough which the photons are propagating has low charge availability(as in slit), there can be very few or no surface plasmons. Also, thepropagation of light is supported along the slit and throughput shouldbe higher for an array of slits versus an array of circular holes of thesame area. Work done on slits much smaller than the transmittedwavelength (32 nm slit) [see Astilean, Lalanne and Palamaru “Lighttransmission through metallic channels much smaller than the wavelength”Optics Communications 175 265-273 March 2000 ] in optically thick metalfilm shows peak in the NIR and visible transmission versus incidentwavelength curves with maxima in the order of 80% efficiency for theplate with a grid spacing of 900 nm. For the strongest peak, 1.183 μm,this is an extraordinary amount in that almost 10 times the amount oflight impinging on the slits is transmitted through them. Also reportedare slits of 10 nm widths, which when excited at resonance, achieve 10%efficiency. Astilean et al. conclude that the resonance condition is notonly a function of the SP resonance but that the metallic wall liningsof the slits act as Fabry-Perot cavities and that greatly enhancedtransmissions occur when the slit satisfies the Fabry-Pérot resonancecondition [see Born, M. and Wolf, E. Principles of Optics. CambridgeUniversity Press 6th ed. 1980 p. 326] with an effective index ofrefraction which depends strongly on the slit width and material.

FIG. 20 shows still another configuration which utilizes the principlesof the present invention. In this arrangement, the light barrier iscomposed of three different materials: a “good” metal layer 160 over asubstrate consisting of an insulator 162 sandwiched between two layersof “bad metal” 164 and 168. As with the other structures, the “good”metal used in layer 160 is one in which the surface plasmons will decayover a relatively long distance as determined by the surface roughnessof the film 160 (which includes the holes) and the relative values ofthe real and imaginary parts of the dielectric function of film 160(where a small imaginary part provides a long delay decay length). Incontrast, the “bad” metal used in the layers 164 and 168 has adielectric function with a large imaginary part so that the surfaceplasmons decay more quickly over a relatively short decay length.

The “bad” metal used in layers 164 and 168 preferably exhibits twoadditional properties which make a significant contribution to thecreation of nanometric light sources. First, the “bad metal” should beopaque to the light emitted from the surface of the “good” metal in thinfilms. Second, the resonance of the “bad” metal layer(s) should beshould be very different than that of the “good” metal. The resonance ofthe metal layers is determined only by the real part of the dielectricfunction for metal, the surface roughness of the metal layers, and thedielectric functions of the materials on either side of the metal layer.

The insulator 162 ensures that there can be no surface plasmoncommunication from top to bottom through bulk plasmons or any otherdirect electronic interaction. Note, however, that the presence of theinsulator 162 may not required if the bad metal satisfies the criteriaexpressed above; that is, is opaque to light emitted from the good metallayer and has a resonance that is very different from the good metallayer.

For the all of the structures described in connection with FIGS. 4-20,the diameter of the hole should be between about 2 nm and 50 nm. Themetallic film layers should, as noted earlier, be at least skin depth ofthe electronic excitation and may be formed, for example, from gold,silver, aluminum, beryllium, rhenium, osmium, potassium, rubidium,cesium, rhenium oxide, tungsten oxide, copper or titanium (if employedat the appropriate frequencies). Suitable dielectric and “bad metal”substrate materials include germanium, silicon dioxide, silicon nitride,alumina, chromia, some forms of carbon and many other materialsincluding some of the metals listed as “good metals” at the appropriatefrequencies. The aperture array with sub-wavelength holes may befabricated using available focused ion beam (FIB) milling techniques.

The physical structures for producing very small spot and slitillumination may be used to advantage in a number of differentapplications as next described.

Optical Data Storage Using Small Spot Illumination

FIG. 21 illustrates the manner in which a nanometric light source arrayof the type contemplated by the invention may be used to increase thestorage density in an optical storage device. The optical memoryconsists of a light source 231, such a solid state NIR laser as shown inFIG. 21. The light from the source 231 is directed onto the metallicfilm surface of a nanometric hole array 235 using a fold mirror 233. Thenanometric hole array 235 collects and funnels the light such that anarray of discrete areas of illumination are directed toward the opticalstorage medium 237. At each area of illumination, a data value stored atthat location in the storage medium controls the intensity of the lightwhich passes to a pixel location on a charge coupled device array (CCD)239 and hence controls the output data value from that CCD pixel. Theholes in the array 239, the data storage regions in the medium 237, andthe pixel locations in the CCD 239 are equally spaced so that they areproperly aligned. A translation mechanism effects movement of thestorage medium relative to the hole array in incremental steps, witheach step distance being equal to the aperture size.

In the year 2000, commercially available CCD arrays have pixel sizes nosmaller than (4 μm)². If this is a limiting case, optics between thestorage medium and the CCD array could be used to allow less movement.The step size would then be down to that demanded by the Rayleighcriterion.

Note also that the amount of data stored at each pixel location may beincreased by storing more than two signal levels; for example, grayscale or color values may be stored as analog signal magnitudes at eachstorage location.

The data reading technique employed in the optical data storage systemis illustrated in FIG. 35. The optical medium 240 is illuminated by thespot illumination from the SPEI array 241 and the light transmissionthrough the medium 240 is read by the radiation detector 243 which maytake the form of a charge coupled device (CCD) array, a complementarymetal oxide semiconductor (CMOS) array, or other array of radiationsensing elements which senses the previously written state of theoptical storage medium at each pixel location.

An alternative optical data storage system using SPEI is shown below inFIG. 36. The system employs semiconductor lasers seen at 245 and 246.The laser 245 is fitted with a write mask 247 and the laser 246 isfitted with a read mask. Both masks are SPEI arrays that provideapproximately 10,000 apertures each 10-50 nanometers in diameter. Theoptical medium seen at 250 rotates or otherwise moves with respect tothe CCD or CMOS detector array seen at 252. The detector array 252 maybe a 100×100 read array, or larger, to provide fast data access.Operating under the control of a CPU 261, a write format processor 263accepts data to be stored and drives a translation system 266 whichmoves the write head comprising the laser 245 and the write mask 247.When the data is read from the storage unit, it is collected in parallelby the detector array 252, multiplexed at 264 and returned to the CPU261.

To achieve a rugged, compact system, the SPEI mask (247 or 248) may befabricated onto the semiconductor or LED light source (245 or 246). Thewrite head (laser 245 and mask 247) may be performed in parallel, but ata different level of parallelism as is achieved in reading. It requiresa higher illumination intensity to write data into the optical medium250 than to read previously stored data due to the need to produce thephotochemical change required for writing at an adequate rate. Toachieve that increased intensity, the SPEI is modified in the mannerdiscussed below in connection with FIGS. 23, 24 and 34. For writing all,only selected central apertures pass through the SPEI array. Atpositions surrounding each central aperture, areas of surface roughness(dimples) deeper than the skin depth of the good metal are positioned asshown in FIGS. 23 and 34, or the central aperture is surrounded by anannular groove as shown in FIG. 24. This technique allows theextraordinary transmission to be retained while only providing emissionfrom the central aperture. This central aperture then becomes thescanned element that is used to write to the medium. This writingfeature can also be used for reading.

Two factors determine the data packing density that can be attainedusing SPEI data storage: the size of the apertures and the lighttransmission fraction achieved.

Cylindrical holes produced using a focused ion beam (FIB) are typicallylimited to an aspect ratio of 5-6:1 for the depth versus diameter.Accordingly, for a read or write mask having a thickness is 275 μm, theminimum aperture diameter is approximately 55 nm. By using thinner Si₃N₄membranes and pushing the limits of the FIB, the ultimate limit isbelieved to be in the vicinity of 10 nm. Devices have been fabricated on150 nm thick silicon nitride membranes. For smaller apertures, stillthinner membranes may be substituted, or the membrane completely may becompletely eliminated. Moreover, the holes need not be cylindrical andmay be tapered and still provide high light transmission.

The light transmission fraction is expected to be proportional to theaperture diameter to the first power. “Shutters” may be placed betweenthe light source (the laser 245) and the SPEI device (the write mask247) to provide parallelism for the write function. The minimum shuttersize may limit the density of emitting apertures. The emission fromselected portions of the SPEI device may be performed using an LCD (notshown (to block the light, or the local dielectric function at theinterface may be alerted as demonstrated by Kim et al. in the paper“Control of optical transmission through metals perforated with subwavelength hole arrays,” Opt. Lett. 24, 256-258 (1999). In still anothershuttering method, conductive wires may be attached to influence theindividual resonant patterns in the device and, thereby, alter theelectron density and the resonance of the surface plasmons in the arealocal to selected aperture in question, thereby modulating theaperture's emission pattern.

The use of SPEI to implement optical data storage systems possessesnumerous important advantages. Using the techniques described above, itis believed that data storage devices capable of storing 2.8 Terabit/in²(with 10 Dm apertures and with the data stored in a binary format) canbe fabricated. SPEI arrays with 50 rn (82 Gigabits/in²) apertures havebeen constructed, and aperture sizes as small as 2 nm are possible topotentially yielding 70 Tb/in² storage densities. As noted earlier, Grayscale or color recording offers the potential for further increases indata density. Data may be read from the device in massively parallelformat, achieving read rates that exceed 1500× those for CD technology.High light transmission fractions (15.3% of the light incident onapertures (50 nm) is transmitted in propagating modes to the opticalmedium) have been achieved in very early devices of SPIE architecture.Because the light is propagating, sub wavelength illumination may beachieved without resorting to near-field techniques. A wide range ofilluminating wavelengths may be employed, ranging from the deepultraviolet to infrared, which permits the selection of a wavelength tooptimize the performance of the photochemical used as the opticalstorage medium. The high light transmission fraction combined withflexibility in the wavelength of the light delivered provide thephotochemist with the possibility of either using existing chemistriesor creating new formulations to take advantage of the properties oflight emitted from SPEI devices. The system operates in ambientenvironments (no cryogenic temperatures or vacuum are required foroperation). SPEI data storage is compatible with a broad range ofapplications to meet the needs of large data centers, high densitybackup system, and storage for desktop and handheld devices. Unlikemagnetic technologies, data stored in a SPEI medium is immune toelectromagnetic impulse.

Surface Plasmon Enhanced Microscopy

FIG. 22 of the drawings illustrates the use of the nanosecond lightsource array as contemplated by the invention to construct a “SurfacePlasmon Enhanced Microscope” (SPEM). A sample 311 is placed between theobjective lens 313 of the microscope and the multi-aperture probe (MAP)315. The sample is mounted on a transparent, flat substrate placed on atranslation stage 321 capable of nanometric movement. The MAP 315 isthen moved into close proximity to the sample 311 and held in place by acompressive force module or proximity sensor 330. In fluorescence mode,light is emitted by a light source, such as a pumped laser, a lightemitting diode, an arc lamp or other white light generator, 340 andtransmitted via neutral density filters 342, polarizers 344, a fibercoupler 346 and an optical fiber 350 down to its terminus at the MAP315, where it is emitted through an array of holes in a mask that hasbeen fabricated onto the end of the optical fiber. The light leaving theholes strikes the sample 311 at its surface. The far field light path358 from the objective 313 passes through a low pass filter 360 to abeam splitter or mirrored shutter at 361 which redirects the light to aarray charge coupled device (CCD) 362 that converts the light intoelectrical signals which are passed to the processor 364 which performsimage capture (frame grabbing) and other image processing functions.

In fluorescence mode, the impinging light is absorbed by fluorophores,which resonate, emitting photons at a different frequency. Thefluorescent light is collected in the far field by the objective lensand then transmitted into oculars 370 or to the data collection device(e.g., the CCD array 362.)

Once the entire sample has been illuminated by the array of apertures,the resulting fluorescence is collected in the far-field. The MAP 315 isthen raised and the sample 311, or the MAP, is indexed to the nextposition and another set of measurements is made. This process isrepeated until the space between the spots, 250 nm to 600 nm, has beenscanned. This is a much easier and faster task than with NSOM. In analternative arrangement the MAP is simply scanned and the raising andlowering steps are eliminated.

It should be clear from the above discussion that it would be difficultto design a probe of the types above with the aim of efficientlytransmitting a multiple of wavelengths chosen to maximize the excitationof a suite of fluorophores. One solution is to make tunable MAPs bydynamically modifying the effective dielectric function of the secondarymetal (the metal probably would be replaced by a semiconductor) duringoperation. By changing the dielectric function of the surface below theprimary metal, the frequency of emission can be changed substantially.[See Kim, T. J., Thio, T., Ebbesen, T. W., Grupp, D. E. & Lezec, H. J.Control of optical transmission through metals perforated withsubwavelength hole arrays.” Opt. Lett. 24, 256-258 (1999) using atwisted-nematic liquid crystal under an array]. It has also been shownthat the application of a magnetic field has strong effects on thedielectric function [see Strelniker, Y. M. & Bergman, D. “Opticaltransmission through metal films with a subwavelenth hole array in thepresence of a magnetic field.” Phys. Rev. B 59, 12763-12766 (1999).Another method of tuning the array may be to have domains surroundingthe apertures in which the density of electrons can be modified bypassing an electric current through that domain. The small capacitanceof the domain would affect the density of the electrons and, hence, theresonance of the surface plasmons.

Multiple MAPs could be constructed with parameters tailored to eachfluorophore of the chosen suite. Each probe would be interfaced to thesample and would present a roughly monochromatic source. As the widthsof the peaks of the resonances of the MAPs will be broad (about 20 nmFWHM), the fluorophores will have to be chosen well with significantdistances between their excitement wavelengths. In this case, the SPEMwill probably be limited to only a few (maybe 6 or so) differentfluorophores. However, the quantum dot offers great promise. Bruchez etal. [“Semiconductor Nanocrystals as Fluorescent Biological Labels”Science 281 1998.] have successfully used quantum dots as biologicalmarkers. Importantly, the quantum dots may be excited by a single sourceand to be multiplexed such that multitudes of dots can be detected andidentified simultaneously.

SPEM has been conceived with clinical and basic research applications inmind and the user interactions have been structured to make it an easytechnique to use. The basic steps, for both clinical and basic researchuse, are:

1. Prepare the sample

2. Select the cells of interest from the slide

3. SPEM automatically captures the data

4. Review the results and generate specific database analyses.

Step 1. Prepare the Sample: In the clinical application the onlyadditional sample preparation step required is to add the antibody-labelreagent to the slide and incubate. The tissue sample preparation stepscurrently in use for pathology slides are done prior to adding the SPEMlabeling reagents (antibody-fluor complexes). Generally for cell culturesamples the cells will be embedded in paraffin and then treated astissue samples for the purposes of preparing them for thermallyconductive substrate, to investigate frozen tissue samples and, undersuitable conditions, it should be possible to study live cells usingSPEM.

Step 2. Select the cells of interest from the slide: With SPEM the userlooks at the slides with a standard far-field microscope prior to thehigh resolution investigation. This allows the user to make use of themorphology data available today and select cells for further analysisthat are the most interesting. To accomplish this, the SPEM system willincorporate a module that allows the user to digitally mark (record thex-y coordinates) the cells for further analysis. This allows the user togather data on different cell types, cells at different stages of thecell cycle, and multiple cells of the same type to increase thestatistical power of the near-field analysis. This also should allow theuser to create multiple slides from the same cell representingsequential cuts from the microtome. The resulting SPEM data can then bereconstructed to create a three dimensional data set of proteinlocations and expression.

Step 3. SPEM automatically captures the data: The SPEM system willexecute the illumination and far-field collection steps described aboveto generate a database of protein localization and expressioninformation.

Step 4. Review the results and generate specific database analyses: Thedatabase created in the previous step provides the user with the abilityto create custom queries to address the biological or clinical questionunder investigation. It is expected that as SPEM matures there would bea library of specific database queries that would be used. Inparticular, for clinical use pathologists would have a set of standardanalyses that are performed with the SPEM to elucidate molecularsignatures of cancer.

SPEM generates a data file consisting of the location of every fluordetected in the cell, and the protein with which it is associated. Thisdata file can be analyzed in a number of ways, including:

i) Generating a map of each protein's location within the cell that issuperimposed on an image of the cell.

ii) Providing the number of copies of each protein that were detected.

iii) Statistics for a number of conditions:

(a) Percentage of copies in the nucleus or cytoplasm

(b) Number of copies of a protein that are within a user specifieddistance of either another protein, or a cellular feature (e.g. cellmembrane)

(c) Comparisons between cells (e.g. mutant and wild type)

(d) Comparisons of protein locations and expression levels between cellsat different stages of the cell cycle.

(e) Comparisons between cells at different developmental levels

iv) Assist in the selection of therapies and determination of prognosesfor cancer patients based on molecular signatures of cancers.

The strengths of SPEM include:

(1) The ability to obtain protein localization and expression data formultiple proteins in a cell from either cell culture or a tissue sample.

(2) Localization resolution better than 75 nm, and possibly as low as 10nm.

(3) Protein expression data based on protein levels, not on mRNA.

(4) Permits the study of low copy number proteins.

(5) Less sensitive to vibrations than NSOM and Atomic Force Microscopy.The level of vibration isolation that is needed is similar to standardmicroscopy techniques.

The MAP used in a SPEM should:

(1) Have an array 75 nm (or smaller) holes that can illuminate a tissuesample with enough energy to excite fluors that have been bound tospecific proteins in the sample.

(2) Have a diameter of at least 20 μm in order to cover a typical cell.

(3) Have the holes in the array spaced far enough apart to permitcollection of optical data from the fluors using far-field optics(greater than the distance imposed by the Rayleigh criterion for theobjective lens being used for collection and the emission wavelength ofthe lowest frequency fluorophore.)

(4) Maintain high resolution registration of the locations of the holesin the array relative to the far-field optics.

(5) Have optical and thermal conductances that are high enough to avoiddeteriorating levels of thermal expansion of the MAP and heating of thesample.

Fabrication of the MAP should be undertaken with the followingparameters in mind: the ability to control aperture size (geometry andthickness); the ability to control aperture spacing; the nature of thematerials (e.g. purity, continuity); and the characteristics of thecoating needed (e.g. continuity and thickness).

In the metal film experiments above, the holes in the films were createdby two methods, both achieving excellent cylindrical geometry. In theSonnichsen experiments, a suspension of polystyrene beads was spin-castonto a very thin (1 nm) adhesive layer on a glass substrate and asubsequent metal film evaporated onto the adhesive and the spheres. Thespheres and the metal covering them were then removed byultrasonification. In the experiments conducted by NEC Research, theholes were created by focused ion beam milling (FIB). This methodallowed more latitude in the hole size and spacing in the metal film.

Because the preferred structures are both heterogeneous and require thatthe hole spacing is uniform (for scanning purposes) or at least wellcharacterized and repeatable from MAP to MAP, the method of spin castingis not useful. FIB can be used but may be expensive for the use of SPEMin clinical settings. Another proposed method of fabrication is to use anaturally occurring structure of alumina. Alumina can be anodicallyetched to produce a uniform nanometric, closely packed honeycombstructure over large areas [see Keller et al. J. Electrochem. Soc. 100411 1953, Thompson et al. Nature 272 433 1978] By usingmicromanipulation, holes could be filled with an insulator or conductorleaving only apertures where desired. The structure would then be coatedwith the chosen electrical conductor and the bottom surface milled awayusing FIB.

The SPEM microscope illustrated in FIG. 22 may be implemented usingcommercially available components. An inverted fluorescence microscopesuch as a Zeiss IM35 or a model from the Zeiss Axiovert family would besuitable for modification. The microscope should have at minimum, twohigh numerical aperture (1.3 or greater) Plan-Apochromat objectives; onefor high magnification (100×) and one for medium magnification (63×)Because the exciting photons are traveling in the MAP, and there is noultraviolet light involved, special glasses and coatings are notrequired. The above objectives have been corrected at the red, green andblue wavelengths for chromatic aberration and will, hence, not be aproblem with different fluorescing colors.

At low levels of fluorescence (low light input is desired to minimizethe effects of photobleaching and possibly, with two-photon excitation,stimulated emission depletion) that may be seen in the SPEM, cooling isrequired when using a charge coupled device (CCD) array to maximizesignal to noise ratio. Zeiss manufactures a suitable high resolution(1300×1030 pixels) thermoelectrically cooled CCD array/frame grabberpackage called Axiocam with color density of 14 bit color classificationwhich is adequate for purposes of multiple fluorescence capture anddiscrimination. The Axiocam is sold by the Microscope Division of CarlZeiss with software called AxioVision that is supplied along with theCCD array, a thermoelectric cooler, frame grabber and image analysissoftware that are integrated with and designed specifically to mate tothe Axio microscopes.

Translation of the sample relative to the MAP and collection opticsrequires a 3 axis translation stage shown generally at 321 in FIG. 22.The step size of the translation stage and its resolution should be lessthan the required resolution desired of the spatial resolution offluorophores in the sample. Mad City Labs (Madison, Wis.) offers such adevice called the Nanobio350. The controller is delivered with LabViewsoftware to make integration with the imaging system easier.

Although the above-noted CCD array is color sensitive anddiscriminating, it is sensitive into the wavelength regime (NIR) of theemission laser. So that the pixels are not saturated with thestimulating radiation and to avoid more computation than necessary, anoptical low pass filter should be placed in the path between the CCDinput and the objective lens of the microscope. There are numeroussuppliers for such filters. If a laser light source is used, a gratingcompensation system may need to be employed to avoid the dispersion thatwould otherwise occur in the fiber. These are available from Coherent.

The current factor that limits the number of proteins that can besimultaneously characterized using SPEM is the limited availability ofspectrally distinguishable fluorophores. Many researchers are working onthis issue and it is expected that SPEM will benefit greatly from theseefforts. Some of the more interesting candidates are described below.

Because the MAP will be designed for efficient transmission of onespecific wavelength of light, a set of fluorophores that can all beexcited by the same wavelength will need to be selected. There are twopromising methods for this: 1) two-photon excitation of fluorescentdyes, using an infrared light source, and 2) quantum dots, using ablue-violet light source. For fluorescent dyes, we would need a set withwell-separated emission wavelengths and narrow spectral peaks. At leasttwo vendors offer products that meet these criteria: Molecular Probes ofEugene, Oreg. offers a set of seven BODIPY dyes, and Amersham PharmaciaBiotech (www.aipbiotech.com) offers a set of five Cy dyes. In addition,new dyes are introduced frequently. Quantum dots are not yetcommercially available for biochemical labeling, but are expected to bein the near future. By tailoring the size of the cavity, quantum dotscan be made with any desired emission wavelength, so conceivably morethan seven could be used within the visible-light spectrum. However,quantum dots are significantly larger than fluorescent dye molecules,10-20 nm vs. 1-1.4 nm effective diameter. This makes fluorescent dyesthe more attractive option. However, if two-photon excitation overheatsthe SPEM probe, quantum dots will be used for the multiple-labelingexperiments.

Quantum dots are nanometer size semiconductor particles withsub-wavelength size pits grown or machined into them. The dimension ofthe pit determines the color of light emitted from a quantum dot. Thepits have dimensions 2 nm (for green light) to 5 nm (for red light), andthe overall particle has a dimension of 10-20 nm. It should be easier todevelop new quantum dots with precisely tuned emission wavelengths(compared to developing a new fluorophore) by tailoring the exactdimensions of the pits in the quantum dots. Quantum dots have a narrowspectral peak width, with a full width at half maximum (FWHM) of 30-35nm [see M. Bruchez Jr., M. Moronne, P. Gin, S. Weiss, and A. P.Alivisatos, “Semiconductor nanocrystals as Fluorescent BiologicalLabels”, Science, 281, 25 Sep. 1998, p. 2013-2016.]. This is comparableto the seven Molecular Probes BODIPY fluorescent dyes, which havespectral peak widths of 22-35 nm FWHM [FIG. 1.2 of Molecular Probes CDhandbook]. Narrow spectral peak widths allow many colors to bedistinguished, allowing many reporters to be used simultaneously.

In addition to fluorescent dyes, and quantum dots mentioned above, othertypes of reporters are also in development. Multiplexing arrangements,which allow a more complex code in each reporter tag, are also indevelopment.

At present, all of these approaches produce tags that are too large.Nanobarcodes (10-20 nm diameter×30 nm long) consist of chips withstripes of reflective gold, silver, and platinum metal. The width andspacing of the lines can be altered. Colloidal particles have been usedto tag beads for combinatorial synthesis [see Battersby B J, Bryant D,Meutermans W, Matthews D, Smythe M L, Trau M, Toward Larger ChemicalLibraries: “Encoding with Fluorescent Colloids in CombinatorialChemistry”, Journal of the American Chemical Society, 122: (9)2138-2139, Mar. 8, 2000]. In this scheme, a 100-micron diameter beadholds multiple 1-micron diameter colloidal particles. Each type ofcolloidal particle holds a unique combination of fluorescent dyes.PEBBLE (Probe Encapsulated By Biologically Localized Embedding) sensorsconsist of fluorescent dyes encapsulated in a polymer matrix; theseparticles can be as small as 20 nm. While these have been used forsensing ion concentrations in cells [see 1 Clark, Heather A; Hoyer,Marion; Philbert, Martin A; Kopelman, Raoul, “Optical Nanosensors forChemical Analysis inside Single Living Cells. 1. Fabrication,Characterization, and Methods for Intracellular Delivery of PEBBLESensors”, Analytical Chemistry, 1999, v.71, n.21, pp. 4831-4836; andClark, Heather A; Kopelman, Raoul; Tjalkens, Ron; Philbert, Martin A,“Optical Nanosensors for Chemical Analysis inside Single living Cells.2. Sensors for pH and Calcium and the Intracellular Application ofPEBBLE Sensors”, Analytical Chemistry, 1999, v.71, n.21, pp. 4837-4843],the technique may be extendable to labeling proteins.

It is possible that the light output from the holes in the MAP willcause illumination of fluorophores or quantum dots in planessubstantially below the surface over which the MAP sits. These moleculescould be excited by the spreading photons and may, therefore, not bedirectly in line with the axis of the holes but could be in between theaxes of several holes resulting in a weak magnitude positive signal atmore than one location, yielding incorrect spatial information andpossibly concentration or color. Methods to reduce this misinformationcould be (but certainly aren't limited) to making the tissue sample orthe image sample as thin as possible or using multi photon excitement.Because of the squared dependence of the two photon excitement oflocation, there will be a substantially higher chance of two photonsarriving concurrently directly in line with the axes of the holes thananywhere else below the MAP, potentially enhancing resolution.

Other modifications to the MAP may be implemented to modify the resonantwavelengths. One method would be to change the in-plane magnetic fieldof the MAP. It has been shown the direction and the magnitude of thefield can dramatically affect the resonant wavelengths by affecting theeffective dielectric functions of the metals. Another method may be tochange the density of electrons in the metals to also affect theeffective dielectric functions. This could be achieved in numerousfashions. The simplest would be simply to “pump” electrons into themetal. Possibly, localization of charges and/or magnetic fields couldallow the MAP to perform read and write operations in storage media andcould be used a polychromatic excitation source for fluorophores.

High Resolution, High Throughput Photolithography

The ability to create spots of light with diameters that are well belowthe wavelength of the light forms the basis of a new approach tolithography and photochemistry. The array structures described above canbe modified in a very simple way to achieve a useful tool forlithography. In the structures discussed in connection with FIGS. 4-20above, all of the apertures in the array penetrate the SPEI lightbarrier and as a result all emit light. For lithography, all but thecentral aperture in a set (the smallest number of apertures required toestablish the resonant condition) would be changed from apertures thatgo through the barrier to elements of surface roughness (dimples orprotuberances) that are deeper than the skin depth and the same diameteras the aperture. Alternatively, the dimples surrounding the centralaperture can be replaced with an annular groove or raised ring having awidth equal to the emitting hole diameter and a depth greater than skindepth. This technique allows the extraordinary transmission to beretained while only providing emission from the central aperture. Thiscentral aperture then becomes the scanned element that is used to writeto the photoresist to perform lithography.

This structure is shown schematically in FIGS. 23 and 24. FIG. 23illustrates a hexagonal pattern of apertures (one emitting aperture 401surrounded by six dimples 403) where the relationship between theresonant wavelength and the spacing is governed by the first equationbelow. Other lattices are permissible with similar equations in whichthe integer indices (i and j in the equations below) are modified forthe specific lattice type (for example a circularly symmetric lattice(central hole surrounded by annuli)) would be governed by the secondbelow equation where p is the radius of 1^(st) annulus and i is aninteger describing the number of annuli away from the center. The thirdequation is for a square array. For a linear array, j is zero.$\lambda_{MAX} = {\alpha_{0}\left( {{{4/3}\left( {i^{2} + {ij} + j^{2}} \right)^{- \frac{1}{2}}\left( {{\left( \frac{ɛ_{1}ɛ_{2}}{ɛ_{1} + ɛ_{2}} \right)^{\frac{1}{2}}\lambda_{MAX}} = {\rho/{i\left( \frac{ɛ_{i}ɛ_{2}}{ɛ_{1} + ɛ_{2}} \right)}^{1/2}}} \right)\lambda_{MAX}} = {{\alpha_{0}\left( {i^{2} + j^{2}} \right)}^{- \frac{1}{2}}\left( \frac{ɛ_{1}ɛ_{2}}{ɛ_{1} + ɛ_{2}} \right)^{\frac{1}{2}}}} \right.}$

where: λ is the wavelength, ∈₁ and ∈₂ are the real portions of thedielectric constants for the metal and the surrounding medium, a₀ is thelattice constant (spacing between dimples/apertures), while i and j areintegers characterizing the particular branch of the surface plasmondispersion. See Raether, Heinz “Surface Plasmons on Smooth and RoughSurfaces and on Gratings” Springer Tracts in Modern Physics v. 111,Springer-Verlag, Berlin 1988.

FIG. 24 shows an alternative arrangement in which the single emittingaperture 407 is surrounded by an annular groove 409 with a width equalto the diameter of the emitting hole. In accordance with the invention,means are employed for limiting the extent of surface plasmon excitationat the exit surface of the emitting hole to the hole itself, or to asmall area surrounding the rim of the hole at its exit, therebyconfining the area of illumination to achieve higher resolution. All ofthe light barrier configurations described above in connection withFIGS. 4-20 may be employed to limit the illumination area produced bythe emitting hole.

The optical system required to execute SPEI lithography is very simple;there are no reduction lenses or steering mirrors. All that is requiredis a somewhat monochromatic light source, such as a filtered broadband(e.g. Hg lamp) source or a laser, the SPEI device, a subnanometertranslation stage (e.g. the nanopositioning systems available from MadCity Labs, Inc. of Madison, Wis.), a proximity sensor to maintain theSPEI device at a proper photoresist distance, and a photoresist coatedwafer.

Three techniques may be used to improve the throughput of the SPEIlithographic process. First, a SPEI device is used to achieve high lighttransmission in order to increase the speed at which the photoresist canbe patterned. The other two approaches increase the parallelism of thewriting operation as described below.

The first level of parallelism is achieved by the creation of a SPEIarray that contains one emitting aperture for each IC on a wafer. Thespacing between emitting apertures will be the same as the spacingbetween ICs on the wafer. By doing this, the same pattern can be writtento all ICs at the same time. To achieve a level of stiffness thatmaintains the flatness of the device and therefore achieves a uniformdevice-to-photoresist spacing, a transmissive substrate may be preparedusing the same techniques used to prepare semiconductor wafers andfabricate the SPEI device on the wafer. The SPEI device should match theindex of refraction of the glass instead of air. The resultingwafer/SPEI device should be rigid enough to allow for a constant CD tobe maintained; otherwise, the SPEI device would have to be farther fromthe photoresist and divergence of the emitted light will increase theminimum CD that can be achieved. If the device is not rigid enough weexpect to fabricate structural elements into it to achieve the desiredstiffness. The light source should provide uniform illumination over thewafer diameter.

The second level of parallelism is achieved by writing multiple featureswithin an IC in parallel. This is achieved with two modifications to thesystem. First, “shutters” are added between the light source and theSPEI device. Second, an SPEI device is constructed that has provides apalette of different shapes. The two basic shapes that would be includedare a circular (or square) aperture and a line segment. Each of theseshapes is preferably provided in different sizes (diameters for thecircular apertures, and lengths and widths for the line segments), andthe line segments preferably have different orientations (horizontal,vertical, +/−45.degree.).

The minimum shutter size will be the consideration that drives thedensity of emitting apertures. Shuttering the emission from portions ofthe device may be performed using a liquid crystal device to block thelight or locally affect the dielectric function of the good metal or byattaching wires to the individual resonant patterns in the device toalter the electron density and, hence, the resonance of the surfaceplasmons in the area local to the aperture in question, therebycontrolling a pattern's emission.

By using the invention to create small illumination spot sizes,lithography employing surface plasmon enhanced illumination providesnumerous advantages, including:

a) small spot size (2-50 nm) for enhanced resolution;

b) high throughput coupled with high resolution, making it particularlyuseful for semiconductor fabrication;

c) high light transmission;

d) no diffraction problems with masks as the critical dimensions and CDsare reduced

e) more flexible range the light wavelengths can be used, deliveringhigh resolution light over a broad range of wavelengths (from deepultraviolet well into the infrared range, supporting development of newphotoresist chemistries for a variety of applications.

f) maskless production technology is compatible with rapid prototypingand low production volumes as well as high volume runs;

g) the cost and complexity of SPEI lithography are compatible withcreation of a system that can be used for rapid prototyping ofsemiconductors, creation of high-resolution masks for e-beam and extremeUV, and other research uses of photolithography;

h) provides a general purpose tool to be used in non-semiconductorlithography applications in the fields of biology, drug discovery, andclinical diagnostics, including lithography applications such asbiosensors, bio-patterning, and array detectors (DNA microarrays,protein and small molecule arrays), all of which that benefit greatlybecause SPEI can deliver small critical dimensions (CDs) withoutresorting to ultraviolet light that damages bio-molecules; and

i) further lithography applications such as MEMS, self-assembly,molecular electronics, and the study of physics phenomena at very smalldimensions.

SPEI photolithography may be employed as a manufacturing method for abinding biosensor or nucleic acid microarray in which the density ofnucleic acid probes substantially exceeds the density that can beachieved using traditional photolithography methods that are limited bythe Rayleigh criterion. SPEI lithography can also be used for any typeof array sensor where photochemistry is used to prepare the surface forimmobilization of a ligand or in situ synthesis of the ligands. Forexample, in very large scale immobilized polymer synthesis systems, asubstrate having positionally defined oligonucleotide probes issynthesized. See, for example, Pirrung et al. U.S. Pat. Nos. 6,416,952;5,143,854; and 5,489,678. In these prior arrangements, conventionalprojection photolithography using masks with UV illumination is used incombination with photosensitive synthetic subunits for the stepwisesynthesis of polymers according to a positionally defined matrixpattern. Each oligonucleotide probe is thereby synthesized at known anddefined positional locations on the substrate. However, the density ofthe array is constrained by the conventional photolithography methodswhose resolution is limited by the Raleigh criterion. By using SPEI,this synthesis process may be performed using a direct write method,eliminating the need to create a mask, and providing significantlyimproved probe density. Direct write is the equivalent of using a paintbrush to paint a picture whereas projection lithography with masks isakin to silk screening the picture. Silk screening, when it iscompatible with the resolution required is faster. However, the masks inphotolithography are expensive and they wear out. This will increasinglybe a serious problem for the semiconductor industry as the feature sizesdecrease the cost of the masks increase and their lifetime decrease.

It will be apparent to one skilled in the art that the use of theinvention for photolithography extends to all photochemical applicationswhere a pattern is created, as photolithography is a specific field ofphotochemistry. This would include the preparation of surfaces forsubsequent operations and/or chemical reactions, or the creation ofmicro- or nano-reaction vessels in which the chemical reaction is causedor promoted or inhibited by the addition of light.

SPEI Applied to Genomics and Systems Biology

Functional genomics and systems biology are fields that address genefunction on tissue or organ system specific bases by studying thecomplex interactions between proteins, RNA, and DNA and otherbiomolecules present in cells and extracellular spaces. To accomplishthis, macromolecules need to be studied in complex mixtures thatreplicate the in vivo environment as closely as possible. The enormityof the task calls for analytical methods that can be scaled to operatecombinatorially, thereby allowing for a genomic scale approach to theproblem. Furthermore, due to the statistical nature of manymacromolecular interactions, it is important to have the ability tostudy them at the single molecule level to avoid the loss of informationresulting from ensemble averaging. This is particularly important insituations with bimodal distributions where the average is notrepresentative of any of the molecules' states. This also imposes therequirement that statistically significant sample sizes be employed toavoid spurious conclusions.

Two leading biological systems that are essential for functionalgenomics and systems biology studies are mixtures of macromolecules insolution (e.g. cell lysates and intact cells.) The value of thesesystems can be enhanced when the capability of studying the impact ofchanges to physical and biochemical environments can be studied in realtime.

The leading tools currently used in functional genomics and systemsbiology are mass spectrometry and multiplexed fluorescent microscopy.Both are powerful tools that have proven to be valuable, but bothrequire some form of sample preparation that make them incompatible withreal time analysis of the impact of changes to the environment of intactcells at the single molecule level with statistically significant samplesizes.

Both confocal microscopy and TIR microscopy suffer from the need tolabel the macromolecules. These labels can interfere with the biologicalfunction of the molecules to which they are labeled. Also, the wash steprequired to remove unbound labels proves problematic when studying thereal time effects of changes to the cellular environment. The problemsassociated with the wash step can be eliminated with the use of GFPfusion proteins; however, these fusions can also alter the biology beingstudied.

In accordance with the present invention, surface plasmon enhancedillumination (SPEI) can be advantageously employed to implement an arraybased technique that can be used to study macromolecules and theirinteractions in solution, and to investigate cell surface phenomena inintact cells. Apparatus using SPEI may be employed to study manydifferent unlabeled macromolecules in parallel. This techniqueidentifies the molecule using signatures that are isolated within a richdata set that is based on the macromolecules' interactions that yieldmeasurable photonics effects or signatures as described below. Thesesignatures are the result of the effects of the interactions occurringat a single aperture, and therefore many signatures can be capturedsimultaneously. This new technique may further incorporate amicrofluidics system to deliver environmental changes to thebiochemistry substrate or the intact cells, thereby allowing the user tocontrol or alter the biochemical and/or physical environment to testtheir hypotheses.

A first arrangement for measuring changes in emitted light when thereare protein or nucleic acid molecules in or near the apertures of a SPEIarray is illustrated in FIG. 25. The molecules 430 approach an aperture433 in the SPEI array 435. The SPEI array 435 is illuminated withbroadband or white light as indicated at 437. A CCD detector 440 detectschanges in the intensity of light transmission through the aperture 433.Alternatively, changes in the local resonant frequency which will changewhat wavelength of light is optimally transmitted through that aperturemay be detected. Electrophoresis or diffusion may be employed to directthe protein molecules into the aperture 433. Alternatively, the devicecan be illuminated with monochromatic light that is scanned across theUV-visible-IR portion of the electromagnetic spectrum and the intensitymonitored as the wavelength is scanned. Changes in the emission spectrafrom the apertures indicate the presence and identity of the moleculesaffecting the changes.

In an alternative arrangement, only a narrow band of light wavelengthswould be monitored to detect specific conditions, such as theconcentration of a particular protein in solution. The front side of theSPEI array may be illuminated with light at a resonant(optimally-transmitted) wavelength for the device, and intensity dataare acquired from the back side of the array for each apertureindividually. When protein molecules are added to the buffer solution,shifts in resonances, or changes in light emission from an aperture, maybe detected as changes in intensity of the light coming through eachaperture individually. For other applications, the SPEI array may beilluminated with light at multiple wavelengths, and the light from eachaperture may be detected at multiple wavelengths.

As a protein molecule 430 approaches the SPEI array 435, then movesthrough an aperture 433 and out the other side, there are five regionswhere data will be collected and analyzed for possible contributions tosignatures that can be used to distinguish between differentmacromolecules. These are shown schematically in FIG. 25 at 451, wherethe molecule is approaching an aperture but not yet in the near field ofthe array 435; at 452 where the molecule is approaching an aperturewithin the near field of the top of the SPEI array 435; where themolecule is within the aperture at 453 (adjacent the “good metal”surface); at 454 adjacent the interior “bad metal” layer; at 455adjacent the dielectric layer; at 456 adjacent the “bad metal” exitsurface of the SPEI array; at 457 where the molecule has left theaperture on the emission side and is still within the near-field of thearray 435; and finally at 458 where the molecule has moved beyond thenear-field of the exit surface of the array.

At each position of the molecule, different effects to be measured,including:

a. Changes from the baseline signal measured when only a buffer solutionis present at the position without macromolecules.

b. Changes of the SPEI emission intensity or resonance shift due tolocal change in index of refraction (more likely when the macromoleculeis not axially aligned with the aperture);

c. Changes in the emission pattern. Since the material on the bottomside of the array is different from the material on the top side, theemission pattern becomes non-symmetric (more likely when themacromolecule is off-axis). Further, the solution on the emission sidecan set up a resonance for surface plasmons in the bottom metal layer,which would cause the emission to become fuzzy, possibly recreating theprolate pattern that was eliminated by adding the second metal. Thepresence of macromolecules at various stages may create measurablescattering of the emitted light.

d. Changes in intensity due to absorption of emitted light.

e. Measurable fluorescence for some molecules.

FIGS. 29 to 32 illustrate the kind of features which may be revealed bythe apparatus described in FIGS. 23-26.

FIG. 29 is an illustration of intensity data which shows the variationof intensity vs. the wavelength of the illumination for a saline bufferat 461, for the buffer with protein at 464, and for the buffer withnucleic acid at 467. Note that FIGS. 29 to 32 are illustrative of themanner in which molecular characteristics may be manifested by intensitydata, and do not reflect actual data.

FIG. 30 illustrates the variation of intensity vs. illuminationwavelength which manifests absorbance changes, with the solid line 472indicating a baseline saline buffer and the dotted line indicating thebuffer with an added macromolecule.

FIGS. 31 and 32 are histograms showing the normalized intensitydistribution of the emitted light from a baseline solution exhibitinglittle or no scatter and intensity distribution data measured when amolecule(s) are added to the solution causing the emission pattern toscatter.

These measurements will be used to extract specific types of informationabout the macromolecules. Measured light scattering and pattern changesmay be used to determine the size, shape and/or orientation of themacromolecules in the solution. Alterations in the SPEI coupling effectmay be measured to indicate the size, shape and orientation of themolecules as well as their dielectric constant. Changes in the intensityof the emitted light may be measured to indicate the size, shape andorientation of the molecules as well as their absorbance andtransmission characteristics. The spectral content of the emissions maybe used to indicate the degree to which the fluorescence of themolecules.

The advantages of SPEI may be utilized in a static (non-flow) system foranalyzing proteins and nucleic acids. As shown in FIG. 26, thearrangement of the stacked combination glass bottom cover 501, an SPEIarray 502 formed from a 150 nm thick silicon nitride membrane, a siliconsupport member 503, and a glass top cover 505. The SPEI array directlyabuts the bottom glass cover plate 501. The silicon support 503 spacesthe top and SPEI array apart by a distance of approximately 200 μm(micrometers) and forms a shaped reservoir 508 200 μm deep and 600×600μm square above the an aperture 510 in the silicon nitride membrane 502.This structure thus forms an enclosed gap on the illumination sidehaving a volume of approximately 68 nL that is used as a fluid reservoirto hold the solution containing the biological macromolecules.

To use the apparatus shown in FIG. 26, the upper surface of the SPEIarray membrane 502 to which the support member 508 is affixed is wettedwith the solution, the array membrane 502 and the support member 503 isset onto cover glass bottom 501, and is then covered with the secondcover glass 505 to prevent evaporation of the sample during the time itis being observed and measured.

A SPEI array may also be used in a system employing means to transportthe cells or molecules to be examined to the array. For example, forcell experiments, the illuminated side of the SPEI array needs to comein contact with, or in very close proximity to, the cells beinginvestigated.

As shown in FIG. 27, the cells 521 may be grown on the surface of atransparent member 523 which is shaped to mate with and be receivedwithin the cavity 527 formed in a silicon support member 529 that isaffixed to the upper surface of a SPEI array 530. The silicon supportmember preferably has the same dimensions as noted above for the supportmember 503 shown in FIG. 26.

An alternative transport structure is shown in FIG. 28 in which an SPEIarray 541 and an attached support member 543 are inverted such that theemission side of the SPEI array is attached to the support frame 541 andthe free side of the SPEI array 541 is illuminated through a transparentmember 545 on which the cells 547 are grown. This structure increasesthe working distance (the path length from the array 541 to thecollection lens (not shown) and thereby decreases the maximum numericalaperture (NA) of the lens system being employed for collection. Thiswill also decrease the resolution (which is inversely related to thenumerical aperture, NA), and also decreases the signal collected ormeasured (which is inversely proportional to the square of the numericalaperture). The decrease in resolution is may be made less problematic ifthe array aperture is surrounded by dimples as shown in FIG. 23 or by anannular groove as shown in FIG. 24. However, when closely-spacedapertures are used, the increased working distance could cause lightfrom adjacent apertures to overlap in the CCD camera image, and thedecrease in transmission may be a problem when the system is beingoperated near the limit of its sensitivity. If either resolution ortransmission is a problem, the structure shown in FIG. 25 may beemployed. A third alternative, reducing the thickness of the supportframe, or to eliminate the support frame altogether, is also possible,but such a construction may be too fragile for some applications.

Applying a voltage across the array will cause charged macromolecules tomigrate through the array (electrophoresis). The rate of migration of amacromolecule in a particular fluid depends on its charge (in thatsolution and at that pH) and its characteristic fluidynamic radius,which determines its drag in that fluid. The conductive metal layers ofthe array will also affect the electric field inside the apertures.Accordingly, the magnitude of voltage applied to create the electricfield should be chosen to move the macromolecules through the array atspeeds compatible with the data acquisition rate.

In some applications, the externally-applied electric field may affectthe behavior of the transmission and resonance characteristics of theSPEI arrays. If the electrical field adversely affects performance, itmay be used to transport the molecules to the desired position, and thenbe shut off the field while making measurements. The electric field maybe generated or applied to cause flow, then the field shut off whileobserving emitted light from each aperture as a function of time. Fastelectrical switching circuitry may be employed bring the field rapidlyto zero, and an oscillating electrical field produced by a waveformgenerator may be used to produce electric fields having differentfrequencies and pulse shapes. By properly applying the motion inducingfield, molecules within the apertures or at other desired states withinthe flow path as enumerated above in connection with FIG. 25.

In a perfusion system, reagents may be delivered in real-time to thecells being studied by micromachining or otherwise effecting fluid pathsinto the cover glass below the array (on the emission side). Themolecules will exit from the perfusion system into a small gap betweenthe bottom cover glass and the array, then move by diffusion through thearray to the cells being studied. Electrophoresis may be used to drawmolecules released from the cells through the array apertures. By havingelectrodes in various locations, and alternately connecting anddisconnecting various pairs, electrophoresis may be employed to drivemolecules through the perfusion system as well. Alternatively, a slightfluid pressure may be applied to the external ports of the microfluidicpassages to cause flow.

The technique described above in connection with FIGS. 25-32 enablessingle cell proteomics, or the study of macromolecules at the singlemolecule detection level for the contents of a single cell. Thesetechniques are particularly useful when the functional genomics datagenerated by the measurement instrument are coupled to physiologicaldata gathered from the cell prior to extracting the cellular contentsfor analysis. In addition, this SPEI analysis technique may be appliedto the study of cell surface phenomena such as the extracellularcomposition of human progenitor cells differentiating. This instrumentmay be used to analyze the composition of the extracellular fluid innear real-time as factors (e.g. EGF, HGF, LIF) are added or removed fromcontact with the cells. This allows the study of cells at the singlecell level to determine the course of differentiation and to improveunderstanding of how it might be controlled. The instrument may be usedby developmental biologists and tissue engineers, and may be employed tothe study of extracellular fluid including the study of the productionof insulin by pancreatic islet cells in response to biochemical stimuli.

The sensor described above can also be used to determine the sizes andconcentrations of proteins in a complex mixture as is currently done ina one dimensional SDS polyacrylimide gel electrophoresis (1D-SDS PAGE)conducted under denaturing conditions. With this approach the size andconcentration information is generated one protein molecule at a time bymonitoring the amount of time that a denatured protein takes to transitone of the apertures in a surface plasmon enhanced illumination (SPEI)device by monitoring the length of time during which the resonance ofthe SPEI device is shifted. By assembling many SPEI apertures inparallel (either single emitting aperture resonant patterns or a fullarray of emitting apertures) the size and concentration data areacquired. This is routinely done in biology laboratories and shifts inbiological research trends will render the gel systems cumbersome asincreasing numbers of samples are analyzed. This invention provides ameans of performing these analyses in an automated and high throughputmanner that is compatible with the increasing numbers of samples to beanalyzed.

It should be further noted that the structures and techniques describedabove in connection with FIGS. 25-32 may also employ apertures formed ina monometallic film as described in connection with FIGS. 1-3, ratherthan a structures in which means are employed for limiting the extent ofelectronic excitation induced in the surface adjacent to the apertureexit. Although the resolution of such monometallic array structures ismore limited, they may be used to implement the biological sensingdevices described where high packing densities are not required.

In accordance with the invention, SPEI can be used to implement abiosensor in which ligands are immobilized at the illuminated surface ofthe SPEI array, and a shift in resonance or other measurable change ismeasured as the ligands' binding partners bind to the illuminatedsurface. As illustrated in FIG. 33, ligands 601 are immobilized at theilluminated surface of an SPEI device indicated generally at 610. Theligands' binding partners, the spores shown at 612, bind to theilluminated surface of the upper “good metal” layer 615 of the device610. Alternatively, the binding partners can be nucleic acids, proteinsand protein complexes, cells, and organisms. As described earlier, theSPEI device further comprises a dielectric 622 sandwiched between “badmetal” layers 624 and 626, with the bad metal layer 624 being adjacentto the illuminated good metal layer 615 and the bad metal layer 626forming the exit surface for illumination passing through the aperture630. The illustrative embodiment of an SPEI biosensor shown in FIG. 33further includes a solid state light source 632, a polarizers 634 forthe incident light which illuminates the surface of the good metal 615,a support (not shown) for the SPEI device 610, and a collection lens andan arrayed detector shown at 635. Note that other sources ofillumination may be used and the light need not be polarized.

The binding effectively alters the electron mobility in the “good” metallayer 615 and changes the resonance condition allowing the light to nolonger be constrained to the condition of exiting from the aperture 630.For small amounts of smaller molecules, such as proteins, the shift inpattern size is somewhat minimal, but a resonance shifts (i.e., changesin the wavelength of peak transmission) may be detected. In addition thebinding of small molecules to proteins, post translational modificationsof proteins, protein-protein interactions, and the binding of nucleicacids can all be detected.

The biosensor illustrated in FIG. 33 preferably employs apertures whichare surrounded by spaced dimples as illustrated in FIG. 23, or by anannular groove as shown in FIG. 24. This technique allows theextraordinary transmission to be retained while only providing emissionfrom the central aperture (seen at 630 in FIG. 33). This centralaperture then becomes the light source that is monitored to detect andquantify the binding events. Alternatively, a square or hex pattern (orany regular geometric pattern) of apertures can be employed. In thisarrangement, independent zones can be established with separationbetween them and either a spectral shift, resonant shift at any of theapertures is indicative of a positive result. The magnitude of the shiftis indicative of the number of binding events and the size of the boundmolecules.

The SPEI array for the biosensor may be constructed using a linear arrayof at least two apertures. For maximum packing density of sets orarrays, polarized light should be used. The polarization directionshould be parallel to the length of the array or set. Using unpolarizedlight does not affect the resonance of the sets or arrays but allowscommunication of surface plasmons in adjacent sets or arrays effectivelymaking the set or array in question substantially larger. As an exampleof a single aperture resonant set employing polarized light, FIG. 34 isprovided. FIG. 34 shows such a set, this minimal set comprises anaperture 651 flanked on each side by dimples 653 and 655, with thesquare aperture 651 and the dimples 653 and 655 being aligned in thedirection of polarization indicated by the arrow 657. The use of squareapertures serves to eliminate variations in the lattice constant byensuring that the aperture-to-aperture spacing is uniform in thedirection of polarization. As in FIGS. 23 and 24, the outside aperturesare dimples that are deeper than the skin depth, thereby contributing tothe resonant effect but not emitting light. The packing density affordedby this geometry is substantially higher than in arrays in which thereis no hole/hole communication (i.e. an array of through holes). Theconfiguration of FIG. 33 also increases the sensitivity of the device.As described above, all of the apertures in this pattern can alsopenetrate the device and emit light, and a shift at any of the aperturesindicates a positive result.

The figures and discussion have described ligands bound to theilluminated side only. It is possible that the ligands could be bound tothe non-illuminated side of the array. In this case, the array spacingof periodic surface features on the illuminated side could be tailoredsuch that dramatic changes in resonance could be seen when the top(illuminated surface) resonated with the bottom (non illuminated)surface. This could be done with several sets of arrays of differinglattice constants so that different concentrations or different targetscould be detected.

Ligands for specific targets of interest (chemical or biological agents,viruses, nucleic acids, proteins and protein complexes, carbohydratesetc.) may be immobilized on the surface of the SPEI device. As thetargets bind with these ligands in the near-field of the metal, theelectron mobility in the metal surface will be altered, thereby changingthe resonant frequencies of that surface and thereby altering thecharacter (spectral transmission and pattern of emission) of the lightemitted from exit surface of the array. The same device and principlescan be used to detect secondary reactions to molecules that have beenbound to the ligands. An important example of secondary reactions is thepost-translational modification of proteins. One can also make use of asecondary reaction to amplify a signal. An example of this amplificationis the use of a secondary antibody or other ligand to the molecules thathave bound to the immobilized ligands. This secondary antibody isconjugated to something (e.g. gold particles) that will increase thechange in the electron mobility of the illuminated surface, thereby“amplifying” the signal.

The same effect is monitored in the commonly used ATR (attenuated totalreflection) surface plasmon resonance instruments (called “SPR”instruments) by measuring the angle at which resonance is establishedwith a fixed wavelength of narrow bandwidth or by varying the wavelengthat a fixed angle. In these instruments, when resonance exists, thenormally “totally” reflected photons are mostly absorbed markingresonance.

The SPEI biosensor of FIG. 33 may be fabricated in different ways toanalyze different sample sizes.

Alternative architectures for the biosensor include the use of a freestanding monometallic film and a monometallic film on a transparentsubstrate. All of the binding and detection methods remain the same.

A small area detector on which ligands will be bound is used to detectsingle molecules, or a very small number of molecules. By increasing thenumber of binding sites, the rate at which the molecule can be detectedin small concentrations increases linearly with the number of sites. Inthis configuration, there are tradeoffs to be made for speed. Thepreviously described multi-layer dielectric/metallic stack shown in FIG.33 allows a very high packing density. It does, however, suffer fromlower transmission than does a monometallic film. While the monometallicfilm may show better transmission, it may not allow maximum packingdensity of patterns. For very large molecules, transmission is not anissue as changes are sensed abruptly with even very low transmissions.For smaller molecules, though, the sensitivity of the collection opticsis the limiting factor and higher transmissions mitigate some of thisdependence. In operation, this arrangement will display behavior similarto a Geiger counter showing a count and a rate.

The second kind of sensor employs a large number of repeating patternsto which ligands for several targets will be immobilized. The ligandsfor any specific compound will be immobilized in several locationsacross the surface of the device to provide for redundant detection ofthe targets.

With repeating patterns, each pattern may comprise two identicalsubpatterns, one of which serves as the active detection area and theother serves as an internal control, providing a baseline of theemission pattern against which the binding results can be compared. Theillumination source may scan through a range of illumination wavelengthsas data is collected.

Since a high density can be achieved with this type of biosensor, manymore repeating patterns may be fabricated than there are targets to bedetected. This allows for some of the density to be deployed to achieveredundancy to enhance the fidelity of the data and to use many differentligands for each compound. The use of different ligands for a compoundenriches the data set in several ways. First, it provides for evenfurther redundancy. Also because different ligands typically havedifferent binding characteristics (sensitivity, linearity, selectivity),a set can be constructed that spans a broader range of sampleconcentration and mixture characteristics. An understanding of thebinding characteristics of the ligands in a set allows for the resultdata to be enhanced by computer processing to improve the fidelity andutility of the information generated in the detection process. This alsomakes the detection more robust in response to mutations, both naturaland engineered, in the targets because it is unlikely that all molecularrecognition sites will be altered.

Both positive and negative controls may be incorporated into thebiosensor design. Negative controls may be provided by immobilizingligands for the targets of interest where the binding capacity of theligands has been eliminated. This provides a raw signal against whichthe positive results can be compared. Positive controls may be providedby immobilizing ligands for a molecule that is not expected to bepresent in the application and drying some of this molecule onto thedevice. This molecule would be solubilized when the sample is added andexpected to bind to its ligand, thereby providing a positive signal toensure proper operation of the device.

To use the biosensor seen in FIG. 33, a sample will be applied to theilluminated surface of the SPEI device and incubated to allow binding tooccur. During this incubation time the emitted light will be monitoredand compared against the internal controls to determine the presence ofthe targets of interest. This detection scheme will provide kinetics ofbinding and concentration for the targets.

A microfluidics system and an aperture configuration that eliminates theneed for a scanned light source may be employed. The microfluidicssystem may perform automated sample preparation and permit theinstrument to perform studies where the effect of changes to thebiochemical composition of the same solution is monitored. Eliminationof the need for a scanned light source can be accomplished by havingeach ligand bound to a set of patterns with differing lattice constants,therefore of different resonant frequencies and with differing loci ofWood's Anomaly. In this arrangement, as the compound binds to itsligands the resonant frequencies of the apertures will shift accordingto the change in mobility of the electrons in the metal surface andaccording to the lattice constants of the different sets. The changesseen in the different sets are measured and compared. Through thecomparison of the changes in the sets' responses to applied compoundsthe amount of bound compounds can be determined.

In an illustrative biosensor whose sensitivity has been optimized bycalculation using a mathematical model of the good metal surface (seeJung et al., Quantitative interpretation of the response of surfaceplasmon resonance sensors to adsorbed films. Langmuir 14, 5636-5648(1998)), a peak resonance of 2 nm was assigned for a positive detectionof the molecules of interest. For maximum sensitivity the followingassumptions were been made in the calculations for large and smallbio-molecules:

1) The molecules of interest are proteins and have an index ofrefraction of 1.6²² and k=0²¹

2) In a solution, in which there are proteins, the proteins are allbound to the ligands (i.e. there exist no unbound proteins and the indexof the solution is equivalent to the index of the solvent by itself

3) Water is assumed to be the solvent at 20 degrees C. (n=1.3345)

4) The height of the solvent layer above bound proteins is zero;immediately above the protein layer is a medium of index of unity. Theheight of the solvent layer adjacent to bound proteins is equivalent tothe thickness of the protein layer

5) The thickness of ligand layer is defined as 10 nm and the index ofrefraction of the ligands is n=1.6, k=0

6) The shape of the resonant device is that shown in FIG. 34.

7) The entire area of the resonant pattern (excluding the hole(s) anddimples (or annuli)) is uniformly weighted as far as collection ofphotons and contribution to surface plasmon resonance

8) The incoming light is polarized and aligned with the dimples

9) The characteristic near-field decay length, I_(d), is λ/4

10) The hole size is 100 nm (smaller holes increase sensitivity)

11) The lattice constant is 500 nm

12) The collector metal (good metal) is aluminum with dielectricfunction according to the Handbook of Optical Constants of Solids (ed.Palik, E.) (Academic Press, Orlando, 1985) at a wavelength ofillumination of 564 nm (the resonance of a clean (ligands at 10 nm witha water layer of thickness 30 nm and air above)

13) The index of “everything else” is unity

14) Protein molecules are assumed to be cubes with characteristicdimension of edge length 30 nm

15) Changes in surrounding indices of reflection are conservativelyassumed not to affect total transmission

For large molecules such as the spores of B. anthracis whosecharacteristic dimension is 300 nm, the sensitivity of the device issuch that one spore yields a change in peak wavelength of 143 nm. Thischange can also be validated by a measurable change in the prolatepattern shape.

For small molecules such as individual proteins whose characteristicdimension is assumed to be 30 nm, the sensitivity of the device is 4protein molecules yielding a change in peak wavelength of 1.91 nm. Ofcourse, smaller molecules can be detected in higher boundconcentrations.

The sensitivity is governed, among other things, by the lattice constantand the size of the hole. Smaller lattice constants and smaller holesboth make for higher sensitivities as both contribute to the area towhich the ligands, and, hence proteins, can be bound. The smaller theratio of the binding area, 2(ρ−s)s (for the pattern in FIG. 9) (where ρis the lattice constant and s is the hole characteristic dimension), tothe characteristic dimension of the protein (φ) and number of proteinmolecules (v),Φ/vΦ², the more sensitive the device will be. Severaltradeoffs must be made when designing these devices. Very thin deviceswill be fragile, while thicker devices, although less fragile, will notallow small holes to be milled. Shorter wavelengths also increase thesensitivity (by allowing smaller lattice constants) but intense UV mayalter the properties of the bio-molecules and affect their binding.

Larger holes than the minimum that can be fabricated may be selected.The smallest distribution of hole sizes is achieved when hole aspectratio is smaller than 4. Narrow distributions of hole size and latticeconstants allow sharper resonances.

The area of the device (and packing and number of resonant patterns) islargely dependent on the minimum volume of solvent that can be dispensedonto the resonant patterns. For minimum sensing times, a minimum ofvolume should be distributed over a maximum area with as close packingof resonant patterns as possible. Also, the collection array or devicethat collects the photons emitted from the resonant patterns should havea very high signal to noise ratio and should be as sensitive aspossible. Because the transmission of the SPEI devices appears to beindependent of irradiance levels, arrayed detectors such as CCDs withoutsingle photon sensitivities can be used by simply increasing theirradiance levels to achieve a satisfactory signal to noise ratio.

It is to be understood that the specific embodiments and applications ofthe invention that have been described are merely illustrativeapplications of the principles of the invention. Numerous modificationsmay be made to the methods and apparatus described without departingfrom the true spirit and scope of the invention.

1. A method for sensing a physical substance, comprising acts of: A)positioning the physical substance in close proximity to a first surfaceof at least one surface plasmon enhanced illumination apparatus; B)irradiating the first surface with electromagnetic radiation; and C)detecting a change in a resonance condition of the at least one surfaceplasmon enhanced illumination apparatus due to the physical substance.2. The method of claim 1, wherein the first surface is an electricallyconductive surface.
 3. The method of claim 1, wherein the at least onesurface plasmon enhanced illumination apparatus includes at least oneaperture that allows at least some of the electromagnetic radiation topass through the apparatus, and wherein the act C) comprises an act of:C1) measuring at least one characteristic of the radiation passingthrough the apparatus.
 4. The method of claim 3, wherein the act C1)comprises an act of: measuring the at least one characteristic of theradiation over a plurality of contiguous radiation wavelengths.
 5. Themethod of claim 3, wherein the act C1) comprises an act of: measuringthe at least one characteristic of the radiation at one or more discreteradiation wavelengths.
 6. The method of claim 3, wherein the act C1)comprises an act of: measuring an intensity of the radiation.
 7. Themethod of claim 3, wherein the act C1) comprises an act of: measuring apeak wavelength of the radiation.
 8. The method of claim 3, wherein theact C1) comprises an act of: measuring a spectrum of the radiation. 9.The method of claim 3, wherein the act C1) comprises an act of:measuring an emission pattern of the radiation.
 10. The method of claim1, wherein the physical substance includes a fluid, and wherein the actA) comprises an act of: applying the fluid to the first surface.
 11. Themethod of claim 10, wherein the fluid is a buffer solution.
 12. Themethod of claim 1, wherein the physical substance includes a fluid, andwherein the act A) comprises an act of: flowing the fluid across thefirst surface.
 13. The method of claim 12, wherein the fluid is a buffersolution.
 14. The method of claim 1, wherein the physical substanceincludes at least one macromolecule.
 15. The method of claim 1, whereinthe physical substance includes at least one small object.
 16. Themethod of claim 15, wherein the act A) comprises an act of: applying afluid containing the at least one small object to the first surface. 17.The method of claim 15, wherein the act A) comprises an act of: flowinga fluid containing the at least one small object across the firstsurface.
 18. The method of claim 1, wherein the act A) comprises an actof: A1) binding the physical substance to the first surface.
 19. Themethod of claim 18, wherein the act A1) comprises acts of: immobilizingat least one ligand on the first surface; and binding the physicalsubstance to the at least one ligand.
 20. The method of claim 19,wherein the at least one surface plasmon enhanced illumination apparatusincludes at least one aperture that allows at least some of theelectromagnetic radiation to pass through the apparatus, and wherein theact C) comprises an act of: C1) measuring at least one characteristic ofthe radiation passing through the apparatus.